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Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis
Open accessAbstract
A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.
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Citations
Cited by
The sequence of sequencers: The history of sequencing DNA
Heather, J. M., & Chain, B. (2016) · Genomics · doi:10.1016/j.ygeno.2015.11.003 · Cited by 1,397
“Both of these improvements contributed to the development of increasingly automated DNA sequencing machines [27,28,29,30,31,32,33], and subsequently the fi rst crop of commercial DNA sequencing machines [34] which were used to sequence the genomes of increasingly complex species.”
BackgroundNext-generation DNA sequencing techniques
Ansorge, W. J. (2009) · New Biotechnology · doi:10.1016/j.nbt.2008.12.009 · Cited by 993
Self-citationSequencing technologies and genome sequencing
Pareek, C. S., Smoczynski, R., & Tretyn, A. (2011) · Journal of Applied Genetics · doi:10.1007/s13353-011-0057-x · Cited by 868
A System for Rapid DNA Sequencing with Fluorescent Chain-Terminating Dideoxynucleotides
Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., & Baumeister, K. (1987) · Science · doi:10.1126/science.2443975 · Cited by 855
Rootwelt, H., Chou, J., Gahl, W. A., Berger, R., Coşkun, T., Brodtkorb, E., & Kvittingen, E. A. (1994) · Human Genetics · doi:10.1007/bf00201558 · Cited by 23
Metadata
- Authors
- Ansorge, W., Sproat, B., Stegemann, J., Schwager, C., & Zenke, M.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 1987
- Volume
- 15
- Issue
- 11
- Pages
- 4593-4602
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2125962952
- Cited by
- 328
- References
- 0
- FWCI
- 22.38
- Type
- journal article
- Language
- en
- Open access
- green
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- Ansorge, W., Sproat, B., Stegemann, J., Schwager, C., & Zenke, M. (1987). Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis. Nucleic Acids Research. https://doi.org/10.1093/nar/15.11.4593
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